The T lymphocyte compartment of the immune system can be divided into a variety of cell subsets. For example, CD4+ T cells represent the T helper cell subset, whereas CD8+ T cells represent the cytotoxic T cell subset. Additionally, CD4+ T helper cells mature into distinct subpopulations that produce different panels of cytokines: the T helper type 1 (Th1) subset produces interleukin-2 (IL-2), interferon-xcex3 (IFN-xcex3) and tumor necrosis factorxcex2, (TNF-xcex2), whereas the T helper type 2 (Th2) subset produces interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) and interleukin-10 (IL-10). The Th1 and Th2 subsets also have differing functional activities. Th1 cells are involved in inducing delayed type hypersensitivity responses, whereas Th2 cells are involved in providing efficient xe2x80x9chelpxe2x80x9d to B lymphocytes and stimulate production of IgG1 and IgE antibodies. For a review of Th1 and Th2 subsets, see Seder, R. A. and Paul, W. E. (1994) Ann. Rev. Immunol. 12:635-673.
Cytokines are thought to play a dominant role in controlling the differentiation of T helper precursors (Thp) to either the Th1 or Th2 lineage. Th 1-associated cytokines, such as IFN-xcex3, can enhance the development of Th1 cells and inhibit the development of Th2 cells, whereas Th2-associated cytokines, such as IL-4 and IL-10, can enhance the development of Th2 cells and inhibit the development of Th1 cells. Thus, cytokines can reciprocally regulate the development and/or progression of either a Th1 or a Th2 response.
The course of certain disease states is influenced by whether a predominant Th1 response or Th2 response is mounted. For example, in experimental leishmania infections in mice, animals that are resistant to infection mount predominantly a Th1 response, whereas animals that are susceptible to progressive infection mount predominantly a Th2 response (Heinzel, F. P., et al. (1989) J. Exp. Med. 169:59-72; Locksley, R. M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A61). In murine schistosomiasis, a Th1 to Th2 switch is observed coincident with the release of eggs into the tissues by female parasites and is associated with a worsening of the disease condition (Pearce, E. J., et al. (1991) J. Exp. Med 173:159-166; Grzych, J-M., et al. (1991) J. Immunol. 141:1322-1327; Kullberg, M. C., et al. (1992) J. Immunol. 148:3264-3270). Many human diseases, including chronic infections (such as with human immunodeficiency virus (HIV) or tuberculosis) and certain metastatic carcinomas, also are characterized by a Th1 to Th2 switch, with elevated expression of IL-10 (see e.g., Shearer, G. M. and Clerici, M. (1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer, G. M. (1993) Immunology Today 14:107-111; Yamamura, M., et al. (1993) J. Clin. Invest. 91:1005-1010; Pisa, P., et al. (1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci, A. S. (1988) Science 239:617-623). Furthermore, certain autoimmune diseases have been shown to be associated with a predominant Th1 response. For example, patients with rheumatoid arthritis have predominantly Th1 cells in synovial tissue (Simon, A. K., et al. (1994) Proc. Natl. Acad. Sci. USA 91:8562-8566) and experimental autoimmune encephalomyelitis (EAE) can be induced by autoreactive Th1 cells (Kuchroo, V. K., et al. (1993) J. Immunol 151:4371-4381).
Velupillai and Ham (Proc. Natl. Acad. Sci. USA (1994) 91:18-22) have shown that schistosome egg antigen (SEA), which expresses the Lewisx antigen, and conjugates of the Lewisx antigen, can stimulate IL-10 production in vitro by B cells from Schistosoma mansoni infected mice, but not B cells from uninfected mice, suggesting that during the course of S. mansoni infection, the observed Th1 to Th2 shift may results from IL-10 production by B cells induced by SEA. The significance of these observations beyond the particular S. mansoni system used, however, is unclear. For example, this work did not demonstrate whether human immune cells (e.g., human immune cells in the absence of S. mansoni infection) were responsive to Lewis antigen-containing compounds, nor whether cell types other than B cells, such as macrophages or T cells, could produce IL-10 in response to stimulation with compounds comprising a Lewis antigen in the absence of S. mansoni infection. Moreover, this work did not demonstrate whether Lewis antigen-containing compounds could stimulate production of other cytokines that regulate development of Th1 and Th2 responses, such as IL-4 or IL-5. Still further, this work did not demonstrate whether Lewis antigen-containing compounds were capable of modulating immune responses in vivo.
Given the role of either Th1 or Th2 cells in the development or progression of many disease states, methods for influencing whether a Th1 or Th2 response is mounted are desirable for a variety of clinical situations. This invention provide methods for modulating immune responses by modulating the interaction of immune cells with a compound comprising a Lewis antigen such that production by the immune cells of at least one cytokine that regulates development of a Th1 or Th2 response is modulated. In particularly preferred embodiments, the methods of the invention can be used to stimulate an antigen-specific immune response in a subject (i.e., the compounds of the invention can be used as adjuvants) or to inhibit allergic responses in a subject. The methods of the invention further can be applied to other clinical situations, including autoimmune disorders, cancer and infectious diseases.
The invention is based, at least in part, on the discovery that stimulation of a variety of cell types, including human immune cells, T cells, macrophages and non-T, non-B splenic cells with Lewis antigen-containing conjugates results in the production of cytokines that regulate the development of Th1 or a Th2 response. Moreover, it has now been discovered that human immune cells are sensitive to stimulation by Lewisy antigen-containing conjugates, that cells from human allergy patients and cancer patients show responsiveness to Lewis antigens and that even normal human peripheral blood mononuclear cells are responsive to stimulation with Lewis-antigen containing compounds. It further has been discovered that Lewis antigen-containing conjugates can be used to stimulate production of a variety of Th2-associated cytokines, including IL-10, IL-4 and IL-5. It still further has been discovered that administration of Lewis-antigen containing compounds is effective for modulating immune responses in vivo. For example, stimulatory forms of the Lewis-antigen containing compounds of the invention can be administered to a subject to promote an antigen-specific immune response (e.g., antigen-specific IgG responses) in the subject. Alternatively, inhibitory forms of the Lewis-antigen containing compounds of the invention can be administered to a subject to inhibit IgE responses in the subject (e.g., in the treatment of allergies).
In one embodiment, the invention provides an immunomodulatory method comprising contacting a human immune cell with an agent that modulates interaction of a compound comprising a Lewis antigen with the human immune cell such that production by the human immune cell of at least one cytokine that regulates development of a Th1 or Th2 response is modulated. The human immune cell can be, for example, a T cell, a macrophage or a B cell. In another embodiment, the invention provides an immunomodulatory method comprising contacting a macrophage with an agent that modulates interaction of a compound comprising a Lewis antigen with the macrophage such that production by the macrophage of at least one cytokine that regulates development of a Th1 or Th2 response is modulated. In yet another embodiment, the invention provides an immunomodulatory method comprising contacting a T cell with an agent that modulates interaction of a compound comprising a Lewis antigen with the T cell such that production by the T cell of at least one cytokine that regulates development of a Th1 or Th2 response is modulated.
In one embodiment of the immunomodulatory methods of the invention, production by immune cells of at least one cytokine (preferably IL-10 or IL-4) that regulates development of a Th1 or Th2 response is stimulated. In this embodiment, the agent with which the immune cells are contacted preferably is a stimulatory form of a compound comprising a Lewis antigen, such as a compound comprising cross-linked (i.e., multivalent) Lewisy oligosaccharides, Lewisx oligosaccharides, Lewisa oligosaccharides or derivatives thereof (e.g., sulfated, sialylated or sulfo-sialylated forms of these oligosaccharides). The stimulatory compound can be, for example, a conjugate of the Lewis antigen and a carrier molecule (e.g., human serum albumin or polyacrylamide). For stimulating responses by human immune cells, the agent preferably comprises a Lewisy oligosaccharide or a derivative thereof.
In another embodiment of the immunomodulatory methods of the invention, production by immune cells of at least one cytokine (preferably IL-10 or IL-4) that regulates development of a Th1 or Th2 response is inhibited. In this embodiment, the agent with which the immune cells are contacted preferably is an inhibitory form of a compound comprising a Lewis antigen, such as a soluble, monovalent (i.e., non-crosslinked) form of a Lewisy oligosaccharide, a Lewisx oligosaccharide, a Lewisa oligosaccharide or a derivative thereof (e.g., sulfated, sialylated or sulfo-sialylated forms of these oligosaccharides). For inhibiting responses by human immune cells, the agent preferably comprises a Lewisy oligosaccharide or a derivative thereof.
The stimulatory or inhibitory compounds of the invention can be contacted with immune cells in vitro to produce one or more cytokines that regulate the development of a Th1 or Th2 response. After in vitro stimulation, the immune cells can be administered to a subject to influence whether a Th1 or a Th2 response predominates in the subject. Alternatively, a stimulatory or inhibitory compound of the invention can be administered to a subject in vivo such that production of at least one cytokine that regulates development of a Th1 or Th2 response is either stimulated or inhibited, respectively, in the subject, thereby influencing whether a Th1 or a Th2 response predominates in the subject.
In a preferred embodiment, the invention provides a method of stimulating a specific immune response to an antigen in a subject comprising: administering to the subject: (i) the antigen and (ii) a stimulatory form of an agent comprising a Lewis antigen, such that a specific immune response to the antigen is stimulated in the subject. In various embodiments, the agent comprises a Lewisy oligosaccharide or a derivative thereof, a Lewisx oligosaccharide or a derivative thereof, a Lewisa oligosaccharide or a derivative thereof, or a Lewisb oligosaccharide or a derivative thereof. The agent can be administered, for example, intranasally, orally, intravenously, intramuscularly, subcutaneously or mucosally.
In another preferred embodiment, the invention provides a method of inhibiting an IgE response to an allergan in a subject comprising administering to the subject, in the presence of the allergan, an inhibitory form of an agent comprising a Lewis antigen, such that an IgE response to the allergan is inhibited in the subject. In various embodiments, the agent comprises a Lewisy oligosaccharide or a derivative thereof, a Lewisx oligosaccharide or a derivative thereof, a Lewisa oligosaccharide or a derivative thereof, or a Lewisb oligosaccharide or a derivative thereof. The agent can be administered, for example, intranasally, orally, intravenously, intramuscularly, subcutaneously or mucosally. In one embodiment of the method, the agent and the allergan are coadministered to the subject.
Another aspect of the invention pertains to pharmaceutical compositions suitable for pharmaceutical administration. The pharmaceutical compositions of the invention typically comprise a stimulatory or inhibitory agent of the invention (e.g., a compound comprising a Lewis antigen) and a pharmaceutically acceptable carrier. In one embodiment, the composition is formulated to modulate responses by human immune cells. In another embodiment, the composition is formulated to modulate responses by macrophages. In yet another embodiment, the composition is formulated to modulate responses by T cells. In particular embodiments, the pharmaceutical formulation is suitable for administration by a specific route of delivery, such as intranasal delivery or oral delivery. Moreover, the pharmaceutical compositions of the invention can be packaged with instructions for using the pharmaceutical composition for a particular purpose, such as to modulate an immune response, for use as an adjuvant, to modulate an allergic response or to modulate an autoimmune disease.
In a preferred embodiment, a pharmaceutical composition of the invention comprises a stimulatory form of an agent comprising a Lewis antigen and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition as an adjuvant for stimulating a specific immune response to an antigen. In another preferred embodiment, a pharmaceutical composition of the invention comprises an inhibitory form of an agent comprising a Lewis antigen and a pharmaceutical carrier, packaged with instructions for use of the pharmaceutical composition in the treatment of an allergy.
The invention further provides pharmaceutical composition comprising a conjugate of a Lewis antigen, or derivative thereof, and a carrier molecule, for stimulating Th2-associated cytokine production, wherein the conjugate comprises optimized amounts of the Lewis antigen, or derivative thereof, for stimulation of cytokine production. For example, in a preferred embodiment, the Lewis antigen, or derivative thereof comprises at least 10% of the conjugate by weight, more preferably at least 15% of the conjugate by weight, even more preferably at least 20% of the conjugate by weight and even more preferably at least 25% of the conjugate by weight.
Still another aspect of the invention pertains to methods for stimulating production of a Th2-associated cytokine by a cell in vitro. The methods involve contacting a cell in vitro with a stimulatory form of an agent comprising a Lewis antigen such that production of a Th2-associated cytokine by the cell is stimulated. Preferably, the cell is selected from the group consisting of a T cell, a macrophage and a non-T, non-B spleen cell.